INTRINSIC CYTOSOLIC CALCIUM BUFFERING PROPERTIES OF SINGLE-RAT CARDIAC MYOCYTES

Intracellular passive Ca2+ buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 mu M (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler ''1799'' was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+](i)) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+](i) during the pulse. In the range of [Ca2+](i) from 0.1 to 2 mu M, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, K-D, of 0.63 +/- 0.07 mu M (n = 5) and a binding capacity, B-max, of 162 +/- 15 mu mol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of K-D = 0.96 +/- 0.18 mu M and B-max = 123 +/- 18 mu mol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+](i) from 0.1 to 1 mu M during a contraction requires approximately 50 mu M Ca2+ to be added to the cytosol.